WO2008007463A1

WO2008007463A1 - Composition for diluting and storing sperm

Info

Publication number: WO2008007463A1
Application number: PCT/JP2007/000745
Authority: WO
Inventors: Naoto Yamashiro; Kohichi Saze
Original assignee: Nippon Zenyaku Kogyo Co., Ltd.
Priority date: 2006-07-12
Filing date: 2007-07-10
Publication date: 2008-01-17
Also published as: JP5238501B2; JPWO2008007463A1

Abstract

It is intended to provide a composition for diluting and storing sperm which does not contain a natural animal-derived component such as serum and can maintain a sperm activity at an equal or higher level compared with a conventional liquid for diluting and storing sperm. By removing a natural animal-derived component such as serum and adjusting other components and concentration, the composition for diluting and storing sperm which can maintain a sperm activity at an equal or higher level compared with a conventional liquid for diluting and storing sperm was found.

Description

Specification

Semen diluted storage composition

Technical field

[0001] The present invention relates to a composition for semen dilution preservation. More specifically, the present invention relates to a composition for semen dilution storage that does not contain natural animal-derived components such as serum and that can maintain semen vitality comparable to or higher than that of conventional dilution solutions.

Background art

[0002] In recent years, in order to produce excellent livestock meat in the field of livestock production, a method of artificially inseminating a large number of females with male semen having a high meat production ability and reproduction ability has been performed. These artificial inseminations enable low-cost use of high-capacity male semen, prevention of diseases through semen, high levels of reproductive performance, shortening of mating time, and cost reduction. Therefore, the spread of artificial insemination is desired.

In pork meat, pork semen retains its semen vitality (fertility) only for about 2 days if the raw semen remains as it is, and the fertility rate drops rapidly after the 3rd day. In addition, the sperm concentration is lower than that of cattle, a large amount of injected semen is required, and it is extremely vulnerable to low temperature sensitization, so it is not easy to preserve. For this reason, there are very few farms that use artificial insemination for pork.

[0003] Conventionally, as a method for storing semen, there are known a method in which an injection semen is diluted several times with a diluent and then stored in a liquid state, and a method in which the semen is stored frozen in liquid nitrogen at 1960 ° C. For liquid storage, there are a medium temperature storage method around 15 ° C and a low temperature storage method of 5 ° C. The medium temperature storage method is a more general storage method, and the conception rate is good, but the practical storage period is as short as 3 to 5 days. The cryopreservation method can be used for about a week, but the conception rate tends to decrease.

In the case of cryopreservation, the freezing method includes the tablet method (pellet method) and the straw method. When frozen semen is used, fertilization performance is said to be inferior to that of natural mating if semen with a semen vitality of at least 3 5 ++ after thawing is fertilized at a suitable time to females with good estrus. Only However, cryopreservation of swine semen is more difficult than cattle and its practical application is delayed.

[0004] Therefore, in order to preserve pig semen while maintaining semen vitality, various protective agents and storage dilutions have been developed. For example, by using hydrocarbons such as decane, a protective agent (Patent Document 1) that does not impair the sperm's physiological function even after cold shock treatment that rapidly cools sperm to 2 to 3 ° C, lauryl alcohol Physiological functions of sperm and cells can be substantially preserved by containing long-chain alcohols having a carbon number of 10 to 14 such as 10 to 14 and lipids as well as ushi serum albumin (hereinafter referred to as BSA). Cell cold injury prevention liquid (Patent Document 2) and the like. Also, by adding and mixing with A liquid containing sodium citrate, BSA, 3 sodium glycephosphate, etc., and B liquid containing dimethyl sulfoxide (DMSO) and ptylhydroxytoluene (BHT), 5 ° C And a diluted solution for storing swine semen that can be used at both storage temperatures of 15 ° C (Patent Document 3). Then, in order to improve the storage stability of the swine semen stock solution, the swine semen stock solution comprising the prepared diluted base solution and the additive solution for pH adjustment added to the diluted base solution immediately before mixing the swine semen Have also been developed (Patent Document 4).

[0005] However, even these conventionally known dilution solutions for storage have a problem that the period during which the semen vitality of pig semen can be maintained is short. In addition, the serum contained as a component contains proteins with unknown origin of various cytokines, growth factors, hormones, etc., which may change the properties of stored semen and may have adverse effects. there were. And since serum contains different components depending on the lot, there was a problem that a uniform composition for semen dilution storage could not be obtained. Therefore, it is desired to provide a uniform semen preservation solution that maintains semen vitality and is completely free of natural animal-derived components.

Patent Document 1: Japanese Patent Application Laid-Open No. 8-26 90 1

Patent Document 2: Japanese Patent Laid-Open No. 10-2 7 9 4 0 1

Patent Document 3: Japanese Patent No. 3 3 6 4 6 80

Patent Document 4: Japanese Patent No. 3 6 3 6 6 0 9

Disclosure of the invention [0006] The present invention provides a composition for diluting and storing a semen that does not contain a natural animal-derived component such as a serum-derived component and that can maintain a semen vitality equivalent to or higher than that of a conventional diluting solution. Offering is an issue. Another object of the present invention is to provide a method for storing semen using this composition for semen dilution storage.

[0007] As a result of diligent research to solve the above-mentioned problems, the present inventors removed animal-derived components such as serum and adjusted other components and concentrations to obtain animal-derived components. The present inventors have found a composition for semen dilution storage that does not contain components and can maintain semen vitality comparable to or higher than that of a conventional storage dilution, etc., and has completed the present invention. Furthermore, the semen preservation | save method was completed by adjusting a semen using the composition for semen dilution preservation | save of this invention.

The composition for semen dilution preservation of the present invention is characterized in that it is difficult to foam when dissolved and mixed with the original semen and is well mixed with semen. In addition, the semen diluted stock solution using the composition for semen dilution preservation of the present invention is characterized in that the semen concentrated portion hardly precipitates and the dispersibility is good. For this reason, the labor of mixing the stored semen once or twice a day, which was required for conventional storage solutions, can be reduced to about once every three days. These characteristics are also considered to be useful in that the semen dilution preserving composition of the present invention can maintain semen vitality comparable to or higher than that of conventional diluting liquids.

[0008] That is, the present invention relates to a method for storing semen using the following composition for semen dilution storage of (1) to (15).

(1) A composition for diluting and storing a semen containing a protective agent, a saccharide, and a pH adjusting agent, and containing no natural animal-derived components.

(2) The composition for semen dilution storage according to (1) above, further comprising an antioxidant.

(3) The composition for semen dilution storage according to the above (1), further comprising an amino acid.

(4) The composition for semen dilution storage according to any one of the above (1) to (3), wherein the natural animal-derived component is a serum-derived component.

(5) The composition for semen dilution storage according to any one of the above (1) to (4), wherein the protective agent is a water-soluble polymer compound of a high molecular organic polymer. (6) The composition for semen dilution storage according to (5) above, containing 0.2 to 1% by weight of a protective agent.

(7) Sugars are glucose, trehalose, sucrose, and lac! The composition for semen dilution storage according to any one of the above (1) to (6).

(8) The pH modifier contains citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA (ethylenediaminetetraaceticacid), and ash! Hydrogen Na. (1) The composition for semen dilution preservation | save in any one of (7).

(9) The composition for semen dilution storage according to any one of (2) to (8) above, wherein the antioxidant is ascorbic acid, dibutylhydroxytoluene, or vitamin E.

(10) The amino acid is at least one selected from the group consisting of L-cystine, glutathione (reduced form), cystine, L (+)-glutamic acid Na_hydrate and glycine. (9) The composition for semen dilution storage according to any one of (9).

(11) The composition for semen dilution storage according to (9) or (10) above, containing 0.1 to 4.2% by weight of an antioxidant or an amino acid.

(1 2) A composition for semen dilution storage containing the following components (a) to (d):

(a) As a protective agent, a water-soluble polymer compound of a polymer organic polymer,

(b) including glucose as a saccharide,

(c) pH regulators include citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA and bicarbonate Na

(d) As an antioxidant or amino acid, at least one selected from the group consisting of L-cystine, glutathione (reduced), cystine, L (+)-glutamic acid Na hydrate, ascorbic acid, glycine and arginine Includes more than one.

(1 3) The composition for semen dilution storage according to (1 2) above, which contains ascorbic acid as an antioxidant.

(14) The composition for semen dilution preservation | save in any one of said (1)-(13) (15) A method for storing semen using the composition for semen dilution storage according to any one of (1) to (13) above.

[0009] Since the composition for semen dilution preservation established according to the present invention does not contain natural animal-derived components such as serum-derived components, there is no fear of adverse effects on the stored semen and can be obtained at low cost. In addition, the composition for semen dilution storage of the present invention is useful because it can maintain semen vitality comparable to or higher than that of conventional storage dilutions.

Brief Description of Drawings

[0010] FIG. 1 shows the dispersibility of a composition for semen dilution storage (Example 1).

BEST MODE FOR CARRYING OUT THE INVENTION

[001 1] The “semen dilution storage composition” of the present invention is a composition for storing semen, which contains a protective agent, a saccharide, and a pH adjusting agent, while containing a natural animal-derived component. A composition that does not contain. Further, the “semen diluted storage composition” of the present invention refers to a composition containing an antioxidant or an amino acid, but not containing a natural animal-derived component.

The composition for semen dilution storage of the present invention has an appearance that does not change for at least 6 months when stored at room temperature (temperature when stored in a stability test room kept at around 23 ° C) or at 4 ° C. It is preferable that Furthermore, it is preferable that the composition for semen dilution storage of the present invention has little change in pH and osmotic pressure when the powder is dissolved in 1 L of purified water, and the semen vitality by storage does not change. The target for storing semen using the “semen dilution-preserving composition” of the present invention is not particularly limited, but cows, pigs, birds, goats and the like are preferable, and these types are not particularly limited. When used for pigs, any kind of pig is acceptable, but it is preferable to use a breeding pig, and the kind of breeding pig is not particularly limited.

The term “not containing natural animal-derived components” in the present invention means not containing components such as serum-derived components. This is because the effects of natural animal-derived components on stored sperm are unknown and may have adverse effects. Semen dilution maintenance of the present invention It is desirable that the existing composition is composed only of components with a clear origin that does not affect the semen in which the composition is stored. Therefore, it is desirable that the composition for semen dilution preservation of the present invention does not contain any natural animal-derived components.

[0013] The "protective agent" of the present invention protects the head and cap so that various degradation enzymes necessary for fertilization contained in the acrosome in the cap of the sperm head are not deleted. This is to prevent damage to the head.

Commercially available dilutions include sushi serum albumin and chicken egg yolk, etc., as protective agents at research institutions. However, the “protective agent” of the present invention may be any component that can sufficiently protect semen even when these natural animal-derived components are not used at all. For example, methyl cellulose (MC), sodium carboxymethyl cellulose (CMC-Na), polyvinylpyrrolidone (PVP), xanthan gum, sodium alginate and the like can be mentioned. Moreover, the water-soluble compound of a high molecular organic polymer etc. are mentioned.

These are preferably contained in an amount of 0.2 to 10.0% by weight, and preferably 0.2 to 8.1% by weight, in the composition for semen dilution storage. This is because the performance may not be maintained unless it is added within this range. Also, if it exceeds 10.0% by weight, there will be a problem of solubility.

[0014] The "saccharide" of the present invention can be any component that can sufficiently protect semen even when no natural animal-derived component is used. Examples include trehalose, sucrose, lactose, glucose and the like. These 50.4 to 59. It is preferable to contain 9 wt% semen dilution storage composition, 52.5 to 59.5 wt ⁰ / o, especially from 53.0 to 59.5 wt ⁰ / o free or It is desirable that

[0015] As the "pH adjusting agent" of the present invention, any component that can sufficiently protect semen can be used even when natural animal-derived components are not used at all. For example, it preferably contains citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDT A and hydrogen carbonate Na. It also contains hydroxyethyl piperazine ethane sulfonic acid (H EP ES). Also good. These semen dilution storage composition, Kuen tribasic N a dihydrate 1 3.3 to 1 5.4 wt ^_0/0, tris (hydroxymethyl) Aminometan 1 1.2 to 1 2.8 wt ⁰ / o, citrate anhydride 5.1 to 6.6 wt. ⁰ / o, EDTA 4.1 to 6.5 wt.%, hydrogen carbonate Na 1.0 to 2.2 wt. In particular, citrate tri-Na dihydrate 14.0 to 15.2 wt%, tris (hydroxymethyl) aminomethane 11.5 to 12.5 wt ⁰ / o, citrate anhydride 5.9 to 6. It is preferable to contain 4% by weight, EDTA4.8 to 5.2% by weight, and hydrogen carbonate Na 1.5 to 1.7% by weight.

As the “antioxidant” of the present invention, any component that can sufficiently protect semen can be used even when natural animal-derived components are not used at all. For example, ascorbic acid, dibutylhydroxytoluene, vitamin E and the like are preferable.

Similarly, the “amino acid” of the present invention may be any component that can sufficiently protect semen even when no natural animal-derived component is used. For example, L-cystine, glutathione (reduced form), cystine, L (+) — glutamic acid Na hydrate, glycine, L—cystine hydrochloride — hydrate, lecithin, ergotionine and arginine It is preferable that the number is at least one.

These are added to the composition for semen dilution storage of the present invention, L_cystine 0.1 to 4.2% by weight, glutathione (reduced form) 0.1 to 4.2% by weight, cystine 0.1 to 4.2% by weight. , L (+) glutamic acid N a _ hydrate 0.1 to 4.2 wt%, Asukorubin acid from 0.1 to 4.2 weight ^_0/0 glycine 0.1 to 4.2 wt ^_0/0, L cysteine hydrochloride monohydrate 0.1 to 4.2 wt% lecithin 0.1 to 4.2 by weight%, Erugochionein 0.1 to 4.2 wt ^_0/0, and arginine 0.1 to 4.2 It is preferable to include at least one selected from the group consisting of% by weight. In particular, ascorbic acid is preferably contained in an amount of 0.2 to 0.22% by weight as an antioxidant, and L (+) glutamic acid Na_hydrate is contained in an amount of 0.2 to 0.22% by weight as an amino acid. Is preferred. If antioxidants or amino acids are not added within this range, performance may not be maintained, and additions exceeding 0.22% by weight affect pH and osmotic pressure during dissolution. There is a case.

[001 7] The “method for preserving semen” of the present invention refers to mixing an aqueous solution prepared by dissolving the semen dilution storage composition of the present invention in purified water when necessary and mixing semen at a constant temperature. It means saving. In the present invention, a mixture of an aqueous solution of a semen diluted storage composition and semen is referred to as a “semen diluted storage solution”.

The mixing ratio of the semen and the aqueous solution of the semen dilution storage composition varies depending on the number of sperm contained in the semen, but the sperm dilution storage solution contains 0.4 to 100 million semen. It is preferable to mix. When used for artificial insemination, it is preferable to use 60 to 10 OmI of the “semen diluted stock solution” prepared in this way. The storage temperature of the “semen diluted stock solution” may be any temperature as long as the semen vitality can be maintained, but it is 4 ° C to medium temperature (15 to 18 ° C), especially medium temperature (15 ° C). It is most preferable that the temperature is constantly maintained at a medium temperature (15 ° C.) by a thermostat or the like. In addition, antibiotics can be added and stored as necessary.

[0018] The "semen vitality" of the present invention refers to the determination of the survival rate and motility of sperm contained in the raw semen according to the following vitality criteria. Generally, it is said that fertilization results are not inferior to natural mating if semen with a vigor of semen of 60 + + or higher is fertilized at a suitable time in females with good estrus, but survival is comparable to or higher than that of a conventional storage dilution. It is preferable to have rate and mobility. In the determination of semen, such as sushi, there is a dedicated machine that uses it, and those that do not have a dedicated machine, such as pigs, can be determined visually by placing semen on a semen test plate.

[001 9] Criteria for vitality

1. Survival rate is expressed in% (approximately 5% increments).

2. The motility was displayed in five levels: + +10, +10, tens, earth, and one.

+ + +: Extremely active forward movement

+ +: Active forward movement + ： Slow forward movement

Sat: Turn or pendulum movement

-: Non-exercise

Hereinafter, in the present invention, all semen vitality was determined according to this standard.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1

“Development composition 1” and “development composition 2” shown in Table 1 were used as compositions for semen dilution preservation. For comparison, “Dilution Composition 1” and “Dilution Composition 2”, whose formulations were disclosed as pig semen dilutions, were used. The following ingredients were used to adjust each composition.

1. Ingredients

1) BSA (Wako Pure Chemical), MC (Wako Pure Chemical) or CMC—Na (Wako Pure Chemical) was used as a protective agent.

2) Glucose was used as a saccharide.

3) As pH adjuster, citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA, hydrogen carbonate Na and HEP ES were used.

4) Ascorbic acid was used as an antioxidant and amino acid.

[0022] 2. Adjustment

Each composition was prepared by mixing the above component 1 using each weight so as to obtain the composition shown in Table 1. The resulting developed composition was filled into an aluminum bag (1 pack) as a semen diluted storage composition and stored at room temperature (23 ° C) avoiding high temperature and humidity.

At the time of use, 1 packet was added to 1 L of purified water, and it was completely dissolved and used. The aqueous solution of the composition for semen dilution storage after dissolution was used immediately.

[0023] 1

[0024] <Storage performance test of semen diluted storage composition (semen vitality measurement)>

The semen diluted stock solution was prepared by adding each solution in Table 1 to an aqueous solution of semen from domestic pigs (Landlace) so that the sperm power was << 0.5 billion ZmL. Amikacin was added to each semen-diluted stock solution to 10 OmgZL and stored at medium temperature (15 ° C). The semen vitality was examined over time from the beginning to 7 days, and the results obtained are shown in Table 2. The determination of semen vitality was performed according to the above criteria using a semen property inspection board.

[0025] [Table 2]

Result

In “Developed composition 1” and “Developed composition 2”, even when MC was used instead of BSA as a protective agent, there was no change in pH and osmotic pressure measured values. It was considered stable even when stored for a week. Also in semen vitality 1 5 It was confirmed that the best activity was maintained compared to the conventional storage composition for 7 days storage under the condition of ° C. Therefore, it has been shown that the composition for semen dilution storage of the present invention can maintain the same level of semen vitality as conventional storage dilutions and the like even without containing animal-derived components such as BSA.

The semen preservation state of the semen diluted stock solution using “Developed composition 1” prepared above was confirmed. For comparison, “Diluted Composition 1” was used. As shown in Fig. 1, the semen diluted stock solution using "Developed composition 1" used "Diluted composition 1" and was shown to have better dispersibility than the semen diluted stock solution.

In addition, the degree of maintenance of these dispersibility was investigated by measuring the change in turbidity over time from the time of dilution to 10 days later by the absorbance (550 nm), and the results obtained are shown in Table 3. As a result, as shown in Table 3, it was shown that the dispersibility of the semen diluted stock solution using “Developed composition 1” was maintained within 3 days after dilution. Therefore, in the semen diluted stock solution using “Developed composition 1”, the labor of mixing the stored semen once or twice a day, which was required for the conventional stock solution of the present invention, is 3 days. It was suggested that it can be reduced to about once.

[0028] [Table 3] Cloth 3rd 7th 10th 10th Diluted composition 1 -1 5.273 0.153 0.141 0.108 Diluted composition 1 -2 5.282 0.155 0.140 0.109 Diluted composition 1 -3 5.388 0.165 0.146 0.116 Average 5.314 0.158 0.142 0.111 Development composition 1 -1 5.215 0.989 0.151 0.121 Development composition 1 -2 5.323 0.912 0.141 0.115 Development composition 1 -3 5.525 1.021 0.166 0.130 Average 5.354 0.974 0.153 0.122 Example 1

[0029] <Preparation of composition for semen dilution storage>

1. Ingredients In the preparation of the composition for semen dilution preservation, the following ingredients were used in place of the ingredients used in Example 1.

1) Protective agent: At least one selected from the group consisting of high molecular weight polymers such as PVP, xanthan gum and alginic acid was selected.

2) pH adjuster: At least one selected from the group consisting of citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDT A, and hydrogen carbonate Na was used. .

3) Antioxidants, amino acids, etc .: L_cystine, glutathione (reduced), cystine, L (+) — at least one selected from the group consisting of glutamic acid Na hydrate, ascorbic acid and glycine Was used.

[0030] 2. Adjustment

It adjusted by combining the said 1 component and mixing so that it might become the composition for semen dilution preservation | save of the following formulation 1-6 next. The obtained composition for diluting and storing semen was filled in an aluminum bag (1 packet) and stored at room temperature (23 ° C) avoiding high temperature and humidity. At the time of use, 1 sachet was added to 1 L of purified water and completely dissolved to prepare an aqueous solution of a composition for semen dilution preservation.

[0031] By adding the following protective agent, sugar, pH adjusting agent, various antioxidants in each formulation, amino acid, etc., the composition for preserving semen diluted in formulations 1-6 was prepared. Protective agent: CMC_N a 0. 1 to 4.0 g

Sugars: Glucose 23.0-28.0 g

PH adjuster: Cenic acid tri-Na dihydrate 6.9 g, Tris (hydroxymethyl) aminomethane 5.65 g, Cubic anhydride 2.9 g, EDTA 2.35 g, Hydrogen carbonate N a 0. 75 g

Antioxidants, amino acids, etc. in each formulation:

Formula 1 ： Glutathione (reduced form) 0.1 g

Formula 2 ； L (+) —Glutamic acid N a _hydrate 0.1 g

Formula 3 ： Ascorbic acid 0.1 g Formula 4; cystine 0.1 g

Formula 5; L—Sistine 0.1 g

Formula 6; glycine 0.1 g

Example 3

[0032] <Stability test over time of semen diluted storage composition>

1. Properties and standards test

Of the composition for semen dilution storage prepared in Example 2, the composition for semen dilution storage of Formula 6 was filled in an aluminum bag, avoiding high temperature and humidity, at room temperature (23 ° C) or at 4 ° C. Stored for 6 months. The aging stability of the semen diluted storage composition during this storage period was examined by standard tests of the appearance of the semen diluted storage composition and the pH and osmotic pressure characteristics of the aqueous solution adjusted at the time of use.

[0033] 2. 15 ° C storage performance test (semen vitality measurement)

Using the semen dilution storage composition of Formula 2, 0.5 billion Zm of domestic pig (land race) semen in an aqueous solution of the semen dilution storage composition dissolved in purified water as in Examples 1 and 2 Zm The semen diluted stock solution was obtained. In preparing the semen diluted storage solution, gently add 5 m I of the semen diluted storage solution to 5 m I of the original semen with 10 billion sperm I, and after 10 minutes, add the semen diluted storage composition. The aqueous solution was prepared by adding 9 O m I. This semen diluted aqueous solution was stored at medium temperature (15 ° C) for 7 days, and then semen vitality was determined. Table 4 shows the results of property and standard tests and storage performance tests.

[0034]

4

[0035] As shown in Table 4, it was confirmed from this result that the composition for semen dilution preservation showed almost no change in properties and specifications even after storage for 6 months, and semen vitality did not decline. Accordingly, it was shown that the composition for semen dilution storage of the present invention can be stored for at least 6 months, and can be adjusted and used as necessary.

Example 4

The preservation performance of the semen vitality of the composition for semen dilution preservation | save of the prescriptions 1-6 adjusted in Example 2 was investigated. For comparison, a composition using BSA as a protective agent was prepared and used. The composition of the composition used for comparison is shown below.

Comparative composition

Protective agent: B S A 2. 0 g

Sugars: Glucose 26.5 g

PH modifier: Cenic acid tri-Na dihydrate 6.9 g, Tris (hydroxymethyl) aminomethane 5.65 g, Cubic anhydride 2.9 g, EDTA 2.35 g, Hydrogen carbonate N a 0. 75 g

Antioxidants, amino acids, etc .: Ascorbic acid 0.1 g

[0037] The semen vitality storage performance of each semen diluted storage composition is the storage described in Example 1. The same as the performance test (semen vitality measurement). The vitality after dilution of semen and the vitality after storage for 7 days at medium temperature (15 ° C) were examined.

[0038] Results

The vitality at the time of dilution of the semen contained in the semen diluted stock solution using the formulations 1 to 6 and the comparative composition was 80 + +10 to 90 +++. The vitality of these semen after storage for 7 days was approximately 60 + + or more for the semen diluted storage compositions of Formulas 1 to 6 and 70 + + for those with high vitality. On the other hand, those using the comparative composition ranged from 4 5 +10 to 5 5 +++. Therefore, it was confirmed that the composition for semen dilution storage of the present invention can maintain semen vitality comparable to or higher than that of a conventional storage dilution without containing animal-derived components. Industrial applicability

[0039] The composition for semen dilution storage of the present invention is safe and inexpensive, and can maintain semen vitality comparable to or higher than that of a conventional storage dilution, etc. It can be used by fertilizers and pig farmers.

Claims

The scope of the claims

[I] A composition for diluting and storing a semen containing a protective agent, a saccharide, and a pH adjusting agent, and does not contain a natural animal-derived component.

[2] The composition for semen dilution storage according to claim 1, further comprising an antioxidant.

[3] The composition for semen dilution storage according to claim 1, further comprising an amino acid.

[4] The composition for semen dilution storage according to any one of claims 1 to 3, wherein the natural animal-derived component is a serum-derived component.

[5] The composition for semen dilution storage according to any one of claims 1 to 4, wherein the protective agent is a water-soluble polymer compound of a polymer organic polymer.

[6] The composition for semen dilution storage according to claim 5, comprising 0.2 to 10.0% by weight of a protective agent.

[7] The saccharide is glucose, trehalose, sucrose, or lactose.

The composition for semen dilution preservation | save in any one of 1-6.

[8] The pH adjusting agent according to claim 1 to 7, wherein the pH adjusting agent comprises citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, anhydrous quaternary acid, EDTA (ethylenediaminetetraaceticacid) and hydrogen carbonate Na. The composition for semen dilution preservation | save in any one.

[9] The composition for semen dilution storage according to any one of [2] to [8], wherein the antioxidant is ascorbic acid, dibutylhydroxytoluene, or vitamin E.

[10] Amino acids are L—cystine, glutathione (reduced), cystine, L (+)

The composition for semen dilution storage according to any one of claims 3 to 9, which is at least one selected from the group consisting of —glutamic acid Na—hydrate and glycine.

[II] The composition for semen dilution storage according to claim 9 or 10, comprising 0.1 to 4.2% by weight of an antioxidant or an amino acid.

[12] A composition for semen dilution storage comprising the following components (a) to (d):

(b) including glucose as a saccharide, (c) as pH adjusting agent, including citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA and hydrogen carbonate Na,

(d) As an antioxidant or amino acid, L-cystine, glutathione (reduced form), cystine, L (+)-glutamic acid N a _ hydrate, ascorbic acid, glycine, L-cystine hydrochloride monohydrate, It contains at least one selected from the group consisting of lecithin, ergotionine and arginine.

[13] The composition for semen dilution storage according to claim 12, comprising ascorbic acid as an antioxidant.

[14] The composition for semen dilution storage according to any one of [1] to [13], further comprising an antibiotic.

[15] A method for diluting or storing semen using the composition for semen dilution storage according to any one of claims 1 to 13.